Our strategy is to prevent transmission of Lyme borreliosis by interfering with the tick's ability to feed to repletion. The proposed project attempts to identify and characterize a family of anticoagulants used by Ixodes ticks needed to permit feeding on their mammalian hosts. Such anticoagulants are vital to the success of tick feeding and in the transmission on tick borne pathogens. We shall explore the family of Ixodes tick anticoagulants by biochemical separation from tick salivary gland and saliva, and by probing cDNA and genomic libraries with appropriate primer fragments. Previous attempts in our laboratories to isolate tick salivary proteins by probing cDNA libraries with antibodies from animals made tick immune resulted in 40 clones; 22 of which were represented by an anticoagulant that we have called SALP-14. In the present proposal we will probe libraries with primers derived from homologous portions of the clones previously identified in order to characterize what we now recognize as a family of anticoagulants vital to tick feeding and transmission of Lyme borreliosis. We will express and purify the recombinant anticoagulants and characterize their activities. To determine active sites, we will generate deletion mutants of these anticoagulants and identify the smallest region essential for effective catalytic activity, as well as antigenicity. The goal is to interfere with feeding by the immune response directed against the functional epitope(s) of the anticoagulant molecules. Feeding to repletion is essential for ticks to complete their life cycle and for the transmission of the pathogens they harbor.